The diabetic state is known to be associated with pancreatic exocrine dysfunction and with a marked fall in pancreatic amylase levels. Although prior studies, in vivo, have suggested that insulin regulates pancreatic amylase content, it has not been clear whether, in vitro, insulin can regulate amylase synthesis in the exocrine pancreas. This question has been difficult to answer because in vitro studies have been hindered by the lack of a pancreatic preparation that is devoid of connective tissue, endocrine islets, or duct cells. Using a relatively pure acinar cell preparation, isolated pancreatic acini, I have recently determined that insulin, in vitro, stimulates both protein and amylase synthesis in the pancreatic acinar cell of diabetic rats. In the present proposal, I plan to expand on these findings in five ways. First, I will determine whether this short term effect of insulin on amylase synthesis is mediated at the transcriptional or translation level, by using inhibitors of transcription such as Actinomycin D and Cordycepin. Second, since the diabetic state is associated with a rise in pancreatic chymotrypsinogen content, I will use specific immunoprecipitation procedures to determine whether insulin can directly inhibit the synthesis of this enzyme. Third, I will determine whether insulin can alter enzyme content via effects on degradation. Fourth, I will determine the effect of the diabetic state and insulin on multiple enzymes and proteins in the acinar cell by using 2-dimensional gel electrophoresis. Fifth, I will compare the short term effects of insulin on enzyme synthesis with its long term effects, both in-vivo and in-vitro, and determine whether insulin regulates the mRNA levels of amylase and chymotrypsinogen under these various conditions. For this purpose, I will employ both CDNA hybridization and in-vitro translation techniques, and may set up an in-vitro culture system of pancreatic acinar cells.